If you’ve ever been burned by eating food that’s too hot, you’ll appreciate first-hand just how sensitive the lining of the mouth can be. If you have seen any marketing for oral care products, you have likely seen claims as to their benefits. In this post, we’ll be looking at the importance of testing oral care products to make sure they are sufficiently mild for the intended use, and how other claims regarding their benefits can be supported with in vitro tests. 

Our physiology varies between the lining of the mouth cavity (the oral epithelium) and that of the gums (the gingival epithelium). The gums are protected by a cornified protective layer similar to the skin barrier, but in the oral cavity, this is absent, leaving the area more exposed and easily damaged. 

As a result, there are markedly different sensitivities between the oral lining and the gums, so it’s important to consider the right approach when testing for possible irritant reactions. 

A wide variety of acute and chronic exposure scenarios 

A wide range of products and devices come into contact with the mouth area. Some of these – such as toothpastes, mouthwashes and chewing gums – are used by millions of people daily, heightening the probability of exposure by sensitive individuals, who may experience irritation as a result of single or repeat use. Others, including specialist dental formulations and surgical instruments, may only contact the mouth and gums on an occasional basis.  And of course, there are longer term, continuous exposures to items such as dental braces and wires to consider. Overall, a wide variety of acute and chronic exposure scenarios exist. 

Irritation potential forms an important part of the safety assessment process for oral care, personal care, healthcare, pharmaceutical and medical device companies. For many ingredients, human skin irritation data may be available, but due to the physiological differences described above, this data can’t be assumed to translate to the lining of the mouth or gums. While there are no in vitro tests with regulatory approval for human oral or gingival irritation, tissue engineering technology can provide sophisticated yet accessible solutions in a non-regulatory context. 

Human oral and gingival epithelia can be reconstructed in vitro to provide a close simulation of these tissues in real life, in terms of both structure and function. The models are well suited to testing ingredients, formulations or devices using protocol modifications designed to suit the test material and to mimic real-life exposure. 

How in vitro oral and gingival irritation tests work 

The item is applied to the surface of the model and incubated for defined exposure times.  Damage to the tissue is measured using a dye (MTT) that’s converted by healthy cells to a blue product which is measured using spectrophotometry. This method results in an “ET50” value – the time taken to cause 50% cell death in the underlying tissue. 

The ET50 approach has been widely used by industry for many years and applied to a variety of tissue types – including skin and eye – as a reliable indicator of irritation potential. Now available for oral and gingival epithelia, these tests can be used to assess the rank order of irritation potential or mildness of a range of formulations or ingredients. The approach is ideally suited to new product development or benchmarking against competitor products. We build in appropriate controls and benchmarks to the test and are building a reference database to further enhance the interpretation of the ET50 result. 

As well as the ET50 determination, histological changes and cytokine expression can be assessed using these human oral and gingival epithelial models. Additionally, inflammation can be induced within the models to look at protective or anti-inflammatory effects of formulations or ingredients. 

Beyond irritation & mildness testing – developing products & supporting claims 

Tests for irritation & mildness are just one area relating to oral care products where in vitro tests are being used to develop novel products. Beyond demonstrating safety, many products seek to protect, improve, and repair the mouth and gums, and of course require evidence of this for both product development and marketing. 

An example of such an investigation is the effect of products on the barrier integrity of gingival tissue. Using techniques such as TEER (transepithelial electrical resistance) can be used to measure barrier integrity at multiple time points without causing damage to the tissue. The average resistance across the tissue is recorded and used to observe changes in the tissue barrier. Certain dyes can also be used, where reduced barrier integrity allows more dye to enter the tissue model. 

Outside of specifically looking at the tissue barrier, techniques such as PCR (polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) can investigate a huge number of effects which the tissue might be undergoing. By looking for changed RNA or protein products (with PCR & ELISA respectively) produced by the tissue (which may be caused by and irritant for example), we can determine if oral care products have and role in reducing or protecting against this irritant. To support this, advanced microscopy techniques are also capable of showing us the changes occurring in the tissue. 

Using test data in different markets 

Globally, regulations can vary considerably. A product may be classed as a medical device in some regions while being covered by cosmetic regulations elsewhere, so the safety assessment needs to include careful consideration of the anticipated geographical markets. While we’re not covering regulatory aspects in this post, we partner with well-established consultants and can provide bespoke advice if needed. The data obtained from the non-regulatory tests described here may be useful as weight-of-evidence support for some regulatory submissions. 

Major changes in global buying trends are already being seen for personal care products in the wake of COVID-19, but many oral care products are considered essentials in our daily self-care routines at home – a sector that has seen robust sales through the crisis. A recent survey cited Colgate as the most chosen personal care brand in the world, and across all categories of fast-moving consumer goods (FMCG), it even tallied up more shoppers worldwide than Coca Cola, growing by a healthy 7% in the 12 months to October 2019. Oral care products have a vast reach, highlighting the critical need to ensure their high level of safety for those using them daily around the world. 

More information:

XCellR8 oral irritation test

XCellR8 gingival irritation test



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